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Clinical and epidemiological research
Extended report
Association of the TNFAIP3 rs5029939 variant with systemic sclerosis in the European Caucasian population
  1. P Dieudé1,
  2. M Guedj2,
  3. J Wipff3,4,
  4. B Ruiz3,
  5. G Riemekasten5,
  6. M Matucci-Cerinic6,
  7. I Melchers7,
  8. E Hachulla8,
  9. P Airo9,
  10. E Diot10,
  11. N Hunzelmann11,
  12. J Cabane12,
  13. L Mouthon13,
  14. J L Cracowski14,
  15. V Riccieri15,
  16. J Distler16,
  17. O Meyer1,
  18. A Kahan4,
  19. C Boileau3,17,
  20. Y Allanore3,4
  1. 1Université Paris 7, Service de Rhumatologie, Hôpital Bichat Claude Bernard, APHP, Paris, France
  2. 2Laboratoire Statistique et Génome, UMR CNRS-8071/INRA-1152/Université d'Evry Val d'Essonne, France
  3. 3Université Paris Descartes, INSERM U781, Hôpital Necker, Paris, France
  4. 4Université Paris Descartes, Service de Rhumatologie A, Hôpital Cochin, Paris, France
  5. 5Department of Rheumatology and Clinical Immunology, Charité University Hospital, Schumannstr, Berlin, Germany
  6. 6Department of Biomedicine, Section of Rheumatology, Florence, Italy
  7. 7Clinical Research Unit for Rheumatology, University Medical Center, Freiburg, Germany
  8. 8Université Lille II, Médecine Interne, Lille, France
  9. 9Rheumatology and Clinical Immunology, Spedali Civili, Brescia, Italy
  10. 10INSERM U618, IFR 135, CHU Bretonneau, Tours, France
  11. 11Department of Dermatology, University of Cologne, Köln, Germany
  12. 12Université Pierre et Marie Curie, Service de Médecine Interne, Hôpital Saint-Antoine, APHP, Paris, France
  13. 13Paris Descartes Université, Médecine Interne, Hôpital Cochin, APHP, Paris, France
  14. 14INSERM CIC3, CHU Grenoble, France
  15. 15Department of Medical Clinic and Therapy “Sapienza” University of Rome, Italy
  16. 16Department for Internal Medicine 3 and Institute for Clinical Immunology, Friedrich-Alexander-University of Erlangen-Nuremberg, Germany
  17. 17Université Versailles Saint Quentin Yvelines, Laboratoire de Biochimie Hormonale et Génétique, Hôpital Ambroise Paré, AP-HP, Boulogne, France
  1. Correspondence to Professor Yannick Allanore, Service de Rhumatologie A, Hôpital Cochin, 27 rue du Faubourg St Jacques, 75014 Paris, France; yannick.allanore{at}cch.aphp.fr

Abstract

Background TNFAIP3 encodes the ubiquitin-modifying enzyme, a key regulator of inflammatory signalling pathways. Convincing associations between TNFAIP3 variants and autoimmune diseases have been reported.

Objective To investigate the association of TNFAIP3 polymorphisms with systemic sclerosis (SSc).

Methods Three single nucleotide polymorphisms (SNPs) in a set of 1018 patients with SSc and 1012 controls of French Caucasian origin were genotyped. Two intergenic SNPs, rs10499194 and rs6920220, and one located in TNFAIP3 intron 2, rs5029939, were selected. The TNFAIP3 rs5029939 found to be associated with SSc in this first set was then genotyped in a second set of 465 patients with SSc and 182 controls from Germany and 184 patients with SSc and 124 controls from Italy. Pooled odd ratios were calculated by Mantel–Haenszel meta-analysis.

Results The rs5029939 G allele was found to be significantly associated with SSc susceptibility (pooled OR=2.08 (95% CI 1.59 to 2.72); p=1.16×10−7), whereas the rs10499194 and rs6920220 variants displayed no association. Only one of the predicted haplotypes investigated in the French sample was significantly associated with SSc (p=8.91×10−8), and this haplotype was discriminating only in the presence of the rs5029939 risk allele, suggesting that this SNP tags the association signal. The strongest associations of rs5029939 with subphenotypes, having large magnitudes for complex genetic disorders, were observed for diffuse cutaneous SSc (pooled OR=2.71 (1.94 to 3.79), p=5.2×10−9), fibrosing alveolitis (pooled OR=2.26 (1.61 to 3.17), p=2.5×10−6) and pulmonary arterial hypertension (pooled OR=3.11 (1.86 to 5.17), p=1.3×10−5).

Conclusion These results suggest that TNFAIP3 is a genetic susceptibility factor for SSc.

https://doi.org/10.1136/ard.2009.127928

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    Systemic sclerosis (SSc) is a complex multiorgan disease affecting the immune system, the microvascular network and the connective tissue. Only a few aspects of the pathogenesis of this disease are known, from the early inflammatory phase to the fibrosis of cutaneous and internal organ connective tissues. Several genetic polymorphisms have recently been identified as susceptibility markers in studies of large samples including replication.1,–,8 Many of the genes identified are known susceptibility factors for other autoimmune diseases, suggesting that the pathological variants may confer susceptibility to a range of autoimmune diseases.9 These findings are consistent with the hypothesis that multiple autoimmune diseases may display dysregulation of the same pathways. Some of the genetic variants identified were associated solely with susceptibility to SSc, whereas others were also found to affect SSc severity.2 3

    Systemic lupus erythematosus (SLE) is the connective tissue disorder whose underlying genetic factors have been most intensively studied. TNFAIP3 was recently identified as associated with, and linked to, SLE, through genome-wide scan and parent-trio investigations.10,–,12 This association was reported to be strong, and some variants also seemed to influence the phenotype and severity of this disease. Furthermore, a large number of genome-wide association studies carried out in the past 2 years identified TNFAIP3 as a susceptibility locus for multiple autoimmune and inflammatory diseases, providing evidence of association with Crohn's disease, coeliac disease, rheumatoid arthritis, type 1 diabetes and psoriasis.13,–,15

    TNFAIP3 (6q23) encodes A20, a zinc-finger protein required for efficient termination of the nuclear factor (NF)-κB signalling axis downstream from tumour necrosis factor receptor (TNFR), toll-like receptor (TLR), interleukin 1 receptor (IL1R) and NOD2. A20 is a unique dual-function, ubiquitin-editing enzyme that catalyses the deubiquitination of several NF-κB pathway proteins, including TRAF6, RIP1, RIP2 and IKKg/NEMO, through an amino-terminal ovarian tumour domain (OTU). The carboxy-terminal, zinc-finger domain of A20 functions as an E3 ubiquitin ligase, catalysing the K48-linked ubiquitination of substrate proteins, thereby targeting them for proteasome degradation.15 The importance of A20 for NF-κB attenuation is evident in transgenic mice lacking A20 expression, which develop severe inflammation and cachexia, are hypersensitive to both lipopolysaccharide and tumour necrosis factor, and die prematurely owing to uncontrolled systemic organ inflammation.16

    In this study, we tested the hypothesis that genetic variants of the TNFAIP3 gene conferring a predisposition to multiple autoimmune phenotypes also confer susceptibility to SSc. We investigated three single nucleotide polymorphisms (SNPs) of the TNFAIP3 gene in our large cohort of European Caucasians.

    Patients and methods

    Study population and study design

    We performed a large case–control association study, including a replication strategy. The ‘discovery set’ consisted of 1018 patients with SSc and 1012 controls from France. The ‘replication set’ consisted of individuals from Germany (465 patients with SSc and 182 controls) and Italy (184 patients with SSc and 124 controls).17 18 For all patients with SSc, we determined LeRoy's cutaneous subtype19 and carried out a phenotypic assessment, as recommended.20 The control groups consisted of healthy unrelated individuals matched to the patients with SSc for ethnicity (all subjects were of European Caucasian ancestry) and also for sex and age (French controls: 85.2% women, mean age 52.2±8.4 years; German controls: 53.5% women, mean age 58.5±10.6; Italian controls: 82.8% women, 56.1±10.4 years). The study was approved by all the necessary local institutional review boards, and written informed consent was obtained from all subjects. The characteristics of patients with SSc are detailed in table 1.

    View this table:
    Table 1

    Characteristics of the patients with SSc in the ‘discovery’ and ‘replication’ sets

    All patients with SSc were tested for antinuclear antibodies by indirect immunofluorescence (IIF), with HEp-2 cells as the antigen substrate (Antibodies Inc, Davis, California, USA). We systematically checked for antibodies specific to SSc. Anticentromere antibodies were identified on the basis of their distinctive IIF pattern. Anti-topoisomerase I antibodies were determined by counterimmunoelectrophoresis. In assessments of vascular phenotype, precapillary pulmonary arterial hypertension (PAH) was diagnosed by catheterisation, as recommended21 and previously described by our group for SSc.22 Pulmonary fibrosing alveolitis was defined as the presence of typical features on high-resolution computerised tomography of the chest, this procedure being carried out in all patients with SSc.

    Genotyping

    Subjects from the discovery set were genotyped for three tagging SNPs: rs10499194 and rs6920220 at the TNFAIP3 locus, and rs5029939 in intron 2 of the TNFAIP3 gene, which displays the strongest association with SLE. We selected these variants for study on the basis of their reported association with other autoimmune diseases. A competitive allele-specific PCR system (Kaspar Genotyping, Kbioscience, Hoddeston, UK) was used to genotype these three SNPs in this sample.2 3 6 For the replication set, the rs5029939 variant was genotyped with the Taqman SNP genotyping assay allelic discrimination method (Applied Biosystems, Foster City, California, USA). Mean genotype completeness exceeded 99% with both methods, for both patients with SSc and controls, with no differences in allele calling. Accuracy exceeded 99%, as shown by duplicate genotyping of 10% of all samples by the two methods.

    Statistical analysis

    Statistical analysis was carried out with R software (version 2.9.1). We used a type I error rate α=5% for all tests. We tested for deviation from Hardy–Weinberg equilibrium by standard χ2 tests (1 degree of freedom) assessing the differences between observed genotype distributions and expected genotype distributions based on control population allele frequencies. Bonferroni correction was applied to all tests of SNP marker association with the disease (the p value multiplied by n−1 SNP) and to all ‘hypothesis-generating steps’ when comparing the SSc subgroups and control (10 phenotypic subsets). p Values remaining significant after this adjustment for multiple testing are indicated in the tables and identified as Padj in the text. The corresponding ORs were assessed by standard logistic regression analysis, with the most frequent genotype taken as the reference. Haplotype diversity was analysed for the three SNPs, with the expectation–maximisation algorithm, as implemented in the haplo.stats R library. The combined data for the three populations were analysed by (1) calculating the homogeneity of ORs between cohorts by the Breslow–Day and by Woolf Q methods; (2) calculating the pooled ORs under a fixed effects model (Mantel–Haenszel meta-analysis) or a random effects model (DerSimonian–Laird), as appropriate.

    Power calculation

    Power was assessed by a standard non-central χ2 approximation, as previously described.23 Taking into account the expected frequency of the rare allele of rs5029939, the discovery set has a power of 88% for detecting an association between SSc and this TNFAIP3 variant, with an OR of 2.0, at the 5% significance level.

    Results

    Discovery set

    The distribution of the TNFAIP3 genotypes and alleles in the French controls was in Hardy–Weinberg equilibrium (table 2) and was very similar to that previously reported for Caucasian populations (table 2). The case–control association study showed an association between the TNFAIP3 rs5029939 variant and SSc, but no association was found for the other two SNPs (table 2). When we considered subphenotypes, we observed a strong significant difference in TNFAIP3 rs5029939 G allele frequencies between the SSc cases and controls, particularly for the subgroups of patients with fibrosing alveolitis, the diffuse cutaneous subtype of SSc, antibodies against topoisomerase I and PAH (table 2). Weaker differences were seen for patients with SSc with digital ulcerations (n=327, allele G 5.35% vs 3.08%; OR=1.78 (1.16 to 2.72), Padj=0.02), with limited cutaneous disease (allele G 5.54% vs 3.08%; OR=1.84 (1.30 to 2.62), Padj=0.005), with positive anticentromere antibodies (allele G 5.30% vs 3.08%; OR=1.76 (1.17 to 2.65), Padj=0.06). As expected, the three SNPs were not in linkage disequilibrium (LD) (figure 1). Only one of the eight regional block haplotypes defined on the basis of the three tagging TNFAIP3 SNPs investigated was found to be significantly associated with SSc, increasing the risk of the disease (table 3). This TNFAIP3 risk haplotype (A-C-G) was discriminating only in the presence of the rs5029939 risk allele (p=8.91×10−8). A trend toward association was also observed for a second haplotype (G-C-G), which increased the risk of disease and also carried the rs5029939 risk allele (p=0.08) (table 3). Consistent with single-marker analysis, the sum of the frequencies of these two TNFAIP3 risk haplotypes was very similar to that for the rs5029939 risk allele (5.7% vs 5.9%), suggesting that most of the association signal detected was due to the rs5029939 tag. We then investigated the rs5029939 SNP in the replication sample.

    Figure 1
    Figure 1

    Linkage disequilibrium of TNFAIP3 rs6920220, rs10499194 and rs5029939. Linkage disequilibrium as measured by r2. Values are given as numerical values within each box.

    View this table:
    Table 2

    Genotype and allele distributions of the TNFAIP3 SNPs in patients with SSc and controls from the discovery sample

    View this table:
    Table 3

    Predicted TNFAIP3 haplotypes in patients with systemic sclerosis and controls in the French Caucasian population

    Replication set

    The distribution of the TNFAIP3 genotypes and alleles in the two replication sets (minor allele frequency (MAF) 2.5% and 3.7%) was very similar to that in the discovery set (MAF 3.08%) (tables 2 and 4). Furthermore, genotype frequencies were observed to be in Hardy–Weinberg equilibrium in both the German and Italian replication populations. The allelic association with SSc was replicated in the German sample for Topo+ patients with SSc and for the PAH subgroup (table 4).

    View this table:
    Table 4

    Genotype and allele distributions for TNFAIP3 rs5029939 in patients with SSc and controls from the German and Italian Caucasian samples

    Combined populations

    Despite the different geographical origins of the three datasets, a test of combinability carried out by the Breslow–Day method identified no significant differences between them. We therefore carried out a pooled OR analysis with the Mantel–Haenszel test, under a fixed effects model. We found that the TNFAIP3 rs5029939 G allele was strongly associated with susceptibility to SSc (pooled OR=2.08 (1.59 to 2.72), p=1.16×10−7), diffuse cutaneous (dc) SSc (pooled OR=2.71 (1.94 to 3.79), p=5.2×10−9), fibrosing alveolitis (pooled OR=2.26 (1.61 to 3.17), p=2.5×10−6), the presence of anti-topoisomerase I antibodies (pooled 2.31 (1.63 to 3.28); p=2.91×10−6) and PAH (pooled OR=3.11 (1.86 to 5.17), p=1.3×105). These results are detailed in table 5 and illustrated in figures 2 and 3. Intra-cohort analysis showed a trend for association for the TNFAIP3 rs5029939 G allele, in comparisons between patients with SSc-PAH (n=119) and patients with SSc free of PAH (n=1361) (MAF 8.82% vs 5.69%, p=0.05) and between the diffuse cutaneous (n=522) and limited cutaneous forms of the disease (n=1011) (7.18% vs 5.44%, p=0.05).

    Figure 2
    Figure 2

    Forest plot showing the results of Mantel-Haenszel meta-analysis for the populations with systemic sclerosis and controls.

    Figure 3
    Figure 3

    Forest plots showing the results of the Mantel–Haenszel meta-analysis for subpopulations with systemic sclerosis (SSc) and controls. (A) Patients with SSc with antibodies against topoisomerase I; (B) patients with SSc with fibrosing alveolitis; (C) patients with diffuse cutaneous SSc; (D) patients with SSc with pulmonary arterial hypertension.

    View this table:
    Table 5

    Overall genotype frequencies for TNFAIP3 rs5029939 and Mantel–Haenszel meta-analysis under a fixed effects model, for the European Caucasian population

    Discussion

    The aim of this study was to investigate the TNFAIP3 gene as a new genetic cause of susceptibility to SSc. We provide here, the first demonstration that the TNFAIP3 rs5029939 variant has a significant role in susceptibility to SSc. These associations were demonstrated with large samples and by genotyping. The allelic frequencies in the control groups were consistent with expected values, confirming the validity of the results presented.

    TNFAIP3 encodes the ubiquitin-modifying enzyme, A20, a key regulator of inflammatory signalling pathways. Associations between TNFAIP3 variants and SLE have been reported in previous studies, and a recent meta-analysis showed that the strongest association was that for rs5029939.12 The rs5029939 risk allele has also been associated with clinical signs of SLE, with heterozygous carriers of the risk allele having a risk of developing renal or haematological manifestations twice that for homozygous risk allele-negative subjects.12 SNP rs5029939 is located in the second intron of TNFAIP3. Its functional impact is unknown, but this SNP is in almost complete LD (r2=0.99) with the TNFAIP3 exon 3 missense SNP rs2230926. Preliminary data have shown that this coding SNP results in an F127C amino acid change in the OTU domain of the A20 protein, potentially decreasing the efficiency with which this protein attenuates NF-κB signalling. However, additional studies are required to determine whether this effect is influenced by TNFAIP3 rs2230926 genotype.10 24 It is entirely possible that rs5029939 mirrors the effect of rs2230926, but further investigations are required to confirm this. Resequencing of the two TNFAIP3 risk haplotypes carrying the rs5029939 SNP is required to identify the causal variant. Recent studies have suggested that the activation of some inflammatory factors may upregulate fibrotic mediators through TLRs, thereby contributing to SSc pathogenesis.25 It has been shown that TLR engagement leads to A20 induction in macrophages and that A20 is essential for the termination of TLR-induced NF-κB activity and proinflammatory cytokine production. This critical role of A20 seems to be independent of the previously described function of this protein in the termination of TNF-induced NF-κB signalling. A20 also acts as a deubiquitinating enzyme, suggesting a molecular link between deubiquitinating activity and the regulation of TLR signals.26 A20 may thus have a critical role in the regulation of TLRs signals, and this topic must be examined in SSc.

    For SLE, meta-analyses have provided odds ratios of about 2.0 for the rs5029939 risk allele and for other SNPs in LD, approaching the magnitudes reported for the HLA-DR2 or HLA-DR3 alleles, the only risk alleles to date for which ORs exceeding 1.5 and approaching 2.0 are consistently obtained. Our results for SSc are very similar. Indeed, we obtained an OR >2.0 for the association of the risk allele with the disease itself, and the OR values obtained were even higher for the subphenotypes (tables 2 and 4). The recent dissection of HLA class II allelic frequencies in SSc revealed ORs for association similar to those for rs5029939.27 The genetic effect of the rs5029939 SNP in the TNFAIP3 gene is also larger than those of any of the recently identified SSc risk genes, including IRF5, STAT4, OX40L and BANK1.1,–,9 Given the low frequency of the rs5029939G risk allele, this effect is consistent with the ‘common disease, rare variant’ hypothesis of complex genetic disease. These results also confirm that certain genes/loci seem to confer predisposition to multiple immune-related disorders, consistent with the hypothesis that a group of genes confers susceptibility to a broad spectrum of immune diseases.9

    The association of the TNFAIP3 rs5029939 risk allele with the clinical and biological status of patients with SSc remained statistically significant after correction for multiple testing, particularly for patients with the diffuse cutaneous subtype of the disease, anti-topoisomerase I antibody-positive patients, patients with fibrosing alveolitis and patients with PAH. The association with interstitial and vascular lung involvement is of particular importance given the impact of these complications on outcome and survival.28 29 Our results are strengthened by the accuracy of phenotyping, which included CT scans for all patients with SSc and right heart catheterisation in cases of suspected PAH. PAH has been identified as a leading cause of death in SSc and, given its severity and the lack of understanding of its risk factors and pathogenesis, there is an urgent need to identify risk factors for the development of SSc-PAH. The results presented here are thus of great potential importance, as they provide a new genetic marker of SSc-PAH risk. They also raise questions about the possible involvement of the NF-κB pathway in this disease, with inflammatory processes, which are frequently observed in various types of human and experimental pulmonary hypertension, being increasingly recognised as major pathogenic components of pulmonary vascular remodelling.30 However, the significant associations with different clinical features suggest that TNFAIP3 rs5029939 is associated with the disease itself, and determination of its influence on the genetic predisposition to specific disease phenotype will need larger samples.

    Many other SNPs in the TNFAIP3 gene and at the TNFAIP3 locus have been identified as being of potential interest, and further analyses of additional polymorphisms, leading to further haplotype analyses, will therefore be required to investigate the association of these polymorphisms and haplotypes with SSc. Our results were obtained with European Caucasian populations, and further replication studies are required to validate their extrapolation to other populations.

    In conclusion, this report provides evidence for a strong association between TNFAIP3 and susceptibility to SSc, indicating that this gene may be added to the list of genes associated with SSc. TNFAIP3 is also associated with other inflammatory rheumatic conditions, suggesting that this gene may confer susceptibility to multiple autoimmune diseases. Functional studies of these genes and studies of gene–gene and gene–environment interactions are required to determine how these genetic associations lead to specific autoimmune diseases and influence their severity.

    Acknowledgments

    The authors would like to thank Drs Amoura Z, Frances C, (Paris, France), Caramaschi P (Verona, Italy), Carpentier P (Grenoble France), Fajardy I (Lille, France) and Sibilia J (Strasbourg France), for providing SSc samples. The authors thank Dr Benessiano J, Centre de Ressources Biologiques, Hôpital Bichat, Etablissement Français du Sang (Paris), Professor B Grandchamp and Dr N Soufir for DNA from controls.

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    Footnotes

    • Funding This work was funded by Association des Sclérodermiques de France, INSERM, Agence Nationale pour la Recherche (Grant number R07094KS) and was supported by Groupe Français de Recherche sur la Sclérodermie. An unrestricted grant from Wyeth France supported the construction of a DNA bank for controls. The German network for systemic sclerosis was funded by the German Federal Ministry for Education and Research (Grant numbers 01 GM 0310 and 01 GM 0634 to IM, GR, NH and IT).

    • Competing interests None.

    • Ethics approval This study was conducted with the approval of the Paris Cochin Hospital France.

    • Provenance and peer review Not commissioned; externally peer reviewed.